Photodynamic therapy (PDT) is approved or in clinical trials for the local treatment of numerous types of malignancies. In the previous funding cycle, detailed spatial analyses of rodent tumors were performed to define the interaction between PDT and tumor physiological properties, including oxygenation and blood flow. These data demonstrate that PDT outcome can be predicted based on individualized monitoring of tumor blood oxygenation or blood flow and thus serve to confirm the therapeutic relevance of intertumor and intratumor heterogeneity. In the renewal of this application, the structural and functional properties of tissue vasculature (vascular microenvironment) will be studied for their effect on cellular, vascular, and molecular responses to PDT. Studies are designed to test the hypothesis that the vascular microenvironment and its accompanying vasoreactivity are effectors of tumor response to PDT. The ability of vascular microenvironment to affect PDT outcome opens the door to methods of altering vascular response in specific vessel types, such as mature vessels, in order to improve treatment efficacy. Accordingly, PDT will be combined with pharmacological modifiers of mature vascular function in an effort to benefit outcome. In clinical studies, the association between vascular maturity and PDT outcome will be investigated for biopsies of non-small cell lung cancer and ovarian cancer from the patients of PDT clinical trials. The studies proposed herein will be performed using many standard biological assays, as well as custom-designed technologies, of which we have substantial experience. Quantitative image analysis of immunohistochemically-stained sections will be used to define vascular microenvironment and PDT effect on spatial distributions of tumor oxygenation and vascular damage. Noninvasive optical spectroscopy (diffuse optical spectroscopy) will be used to longitudinally monitor tumor physiological processes such as oxygenation and blood flow over the course of treatment. Using these techniques the following specific aims will be addressed: Specific Aim 1. To define how vascular microenvironment affects tumor physiological and cytotoxic responses to PDT. Specific Aim 2. To define how modulation of vasoreactivity during PDT affects tumor and normal tissue responses. Specific Aim 3. To explore the association between vascular microenvironment and therapeutic outcome in patients treated with PDT. PUBLIC HEALTH RELEVENCE: The data generated in this proposal will define the effect of vascular microenvironment on vascular, cellular, and therapeutic responses to photodynamic therapy (PDT), and determine how therapeutic outcome may be benefited by modulation of vasoresponse during illumination. The vascular reactivity of mature blood vessels during PDT will be altered using common clinical pharmacological agents, which makes this approach toward therapy optimization highly clinically relevant. In parallel with the preclinical studies, clinical investigation will address the question of how vascular microenvironment affects patient response to PDT, thus providing necessary groundwork for the design of a clinical trial combining PDT with drugs that alter mature vessel reactivity.